The present invention relates to a hair-growing agent comprising a protein kinase C-specific inhibitor.
There is disclosed that substances with a protein kinase-inhibiting activity, 3-amino/hydroxy-4-[4-benzoyl-phenyl carbonylamino/oxy]azepanes stimulate the growth of hair (EP663393 A1); but said proteinkinase inhibitors have a protein kinase A (hereinafter referred to as PKA)-inhibiting activity along with a protein kinase C (hereinafter referred to as PKC)-inhibiting activity.
In hair-follicle organ culture systems, a PKC inhibitor, H-7 is known to release the hair growth-retarding activity of a PKC-activating substance, 12-O-tetragalloyl-phorbol-13-acetate (British Journal of Dermatology, 133, 5, 686-693, 1995). Said H-7 is further known to have a PKA-inhibiting activity along with a PKC-inhibiting activity (BIO/TECHNOLOGY, 8, 732, 1990).
In hair-follicle organ culture systems, the PKC inhibitor, H-7 releases the hair growth-inhibiting activity of 12-O-tetragalloyl-phorbol-13-acetate, but said PKC inhibitor has not been known to have a hair growth-promoting activity (British Journal of Dermatology, 133, 5, 686-693, 1995).
Protein kinase inhibitors having both a PKC-7inhibiting activity and a PKA-inhibiting activity could not always be expected to produce satisfactory hair growth-promoting results. H-7 and 3-amino/hydroxy-4-[4-benzoyl-phenyl carbonylamino/oxy]azepanes could not always be expected to produce satisfactory hair growth-promoting results, because such compounds have both a PKC-inhibiting activity and a PKA-inhibiting activity.
The present inventors have first found that PKC-specific inhibitors produce satisfactory hair growth-promoting results.
The object of the present invention is to provide a safe and effective ahir-growing agent comprising, as an active ingredient, a PKC-specific inhibitor, and pharmaceutically acceptable vehicles.
The protein kinase C-specific inhibitor as referred to herein is a protein kinase inhibitor having a PKC-inhibiting activity while having a PKA-inhibiting activity as little as possible. Specifically, any inhibitor can be used so long as the ratio of its 50% PKA-inhibiting constant (hereinafter referred to as PKA-IC50) to its 50% PKC-inhibiting constant (hereinafter referred to as PKC-IC50), PKA-IC50/PKC-IC50, is not smaller than 3.0 when determining PKC-IC50 and PKA-IC50 according to the following PKC-inhibiting, activity measuring method and according to the following PKA-inhibiting activity measuring method, respectively. For example, usable herein are protein kinase inhibitors having PKA-IC50/PKC-IC50/PKC-IC50 of from 10 to 109.
For determination of PKA-inhibiting activity of protein kinase inhibitors having a low PKA-inhibiting activity, the following method requires a large amount of such a protein kinase inhibitor to be determined therein, in which, therefore, the protein kinase-inhibiting activities of the protein kinase inhibitor could be measured only up to its uppermost soluble concentration in the system. Accordingly, if such a protein kinase inhibitor with low PKA-inhibiting activity is used, the value of PKA-IC50/PKC-IC50 capable of being numerically determined in said method will be about up to 109. The protein kinase inhibitors usable: in the present invention have its value of PKA-IC50/PKC-IC50 of not smaller than 3, without being defined by its upper value of PKA-IC50/PKC-IC50(109) capable of numerically determined by said method.
Preferred protein kinase inhibitors for use are those having a value of PKA-IC50/PKC-IC50 of from 10 to 109.
(1) Method for Measuring PKC-inhibiting Activity:
To measure the PKC-inhibiting activity of a protein kinase inhibitor, referred to is the Kikkawa et al""s method (Journal of Biological Chemistry, 257, 13341, 1982).
Precisely, 10 xcexcl of a sample to be tested for determining its activity is added to 250 xcexcl of a solution comprising 2.5 xcexcmols of magnesium acetate, 50 xcexcg of histone Type IIIS (produced by Sigma Co.), 20 xcexcg of phosphatidylserine, 0.8 xcexcg of diolein, 25 nmols of calcium chloride, 5 xcexcg of a crude enzyme (as partially purified from a rat brain according to the Kikkawa et al""s method) and 5 xcexcmols of Tris-HCl buffer (pH 7.5), and incubated therein at 30xc2x0 C. for 3 minutes.
After the completion of the incubation, 1.25 nmols of [g-32P]ATP (from 5xc3x97103 to 10xc3x97103 cpm/nmol) is added to the system, and phosphorylation reaction is carried out at 30xc2x0 C. for 3 minutes, and thereafter the reaction is terminated by adding 25% trichloroacetic acid thereto.
The resulting reaction mixture is filtrated through a cellulose acetate membrane (pore size: 0.45 xcexcm) (produced by Toyo Filter Co.), and the membrane is washed four times with 5% trichloroacetic acid, and thereafter the radioactivity of the residue remained on the membrane is measured to be a value of the sample.
On the other hand, the same process as described above is repeated without adding the sample to the system, and the radioactivity is obtained to be a control value.
The molar concentration of the sample having the value of radioactivity which is 50% of the control value is obtained to be the 50% PKC-inhibiting constant (PKC-IC50) of the sample.
(2) Method for Measuring PKA-inhibiting Activity:
To measure the PKA-inhibiting activity of a protein kinase inhibitor, referred to is the Kuo et al""s method (see Biochemistry, 6, 1349, 1969).
Precisely, 10 xcexcl of a sample to be tested for determining its activity is added to 250 xcexcl of a solution comprising 5 xcexcmols of Tris-HCl buffer. (pH 6.8), 2.5 xcexcmols of magnesium acetate, 100 xcexcg of histone Type IIS (produced by Sigma Co.), 0.25 nmols of c-AMP and 200 xcexcg of a crude enzyme (as partially purified from a calf heart according to the Kuo et al""s method), and incubated therein at 30xc2x0 C. for 3 minutes.
After the completion of the incubation, 1.25 nmols of [g-32P]ATP (from 5xc3x97103 to 10xc3x97103 cpm/nmol) is added to the system, and phosphorylation reaction is carried out at 30xc2x0 C. for 3 minutes, and thereafter the reaction is terminated by adding 25% trichloroacetic acid thereto.
The resulting reaction mixture is filtrated through a cellulose acetate membrane (pore size: 0.45 xcexcm) (produced by Toyo Filter Co.), and the membrane is washed four times with 5% trichloroacetic acid, and thereafter the radioactivity of the residue remained on the membrane is measured to be a value of the sample.
On the other hand, the same process as described above is repeated without adding the sample to the system, and the radioactivity is obtained to be a control value.
The molar concentration of the sample having the value of radioactivity which is 50% of the control value is obtained to be the 50% PKA-inhibiting constant (PKA-IC50) of the sample.
Specific examples of the protein kinase C-specific inhibitor for use in the invention may include polymyxin B, calphostin C, palmitoyl-DL-carnitine and hexadecylphosphocholine (militefosine, produced by Sigma Co.), and also their pharmaceutically-acceptable salts.
The pharmaceutically-acceptable salts may include, for example, hydrochlorides, hydrobromides, sulfates, nitrates, formates, acetates, benzoates, maleates, fumarates, succinates, tartrates, citrates, oxalates, methanesulfonates, toluenesulfonates, aspartates, and glutamates.
The hair-growing agent of the present invention may be taken any form, provided that it properly contains the protein kinase C-specific inhibitor of the invention. For example, a liquid or solid hair-growing agent comprising the protein kinase C-specific inhibitor of the invention along with pharmaceutically acceptable vehicles is used.
The liquid or solid hair-growing agent may include liquid-type preparations such as hair liquids, hair tonics and hair lotions; and solid-type preparations such as ointments and hair creams. These agents can be prepared by any ordinary methods, while adding the protein kinase inhibitor of the invention to suitable vehicles.
The amount of the protein kinase C-specific inhibitor to be in the hair-growing agent of the present invention greatly varies, depending on the intensity of its inhibiting activity and also on its endermic absorbability to be derived from its physical properties, but may be generally from 10xe2x88x926 to 10% by weight (hereinafter referred to as %) based on the agent in terms of the content of a single compound of the inhibitor or a mixture of plural compounds thereof.
The preferred vehicles for the liquid-type preparations are those that are generally used in ordinary hair-growing agents, such as pure water, ethanol, polyalcohols, oils and fats. If desired, any additives may be added thereto.
The polyalcohols may include, for example, glycerol, 1,3-butylene glycol, propylene glycol, etc.
The oils and fats may include, for example, wheat germ oil, camellia oil, Jojoba oil, olive oil, squalane, safflower oil, macadamia, nut oil, avocado oil, hydrogenated soybean lecithin, etc.
The additives may include, for example, fragrances, surfactants, microbicides, etc. If desired, any of antioxidants, hormones, ultraviolet absorbents, anti-inflammatory agents, refrigerants, moisturizers, vitamins, herb extracts, tinctures, etc. may be added to the preparations.
As the fragrances, any fragrance usable in ordinary cosmetics, etc. are employable herein.
The surfactants may include, for example, polyoxyethylene(60) hardened castor oil, polyoxyethylene(8) oleyl ether, polyoxyethylene(10) oleyl ether, polyoxyethylene(10) monooleate, polyoxyethylene(30) glyceryl monostearate, sorbitan monostearate, polyoxyethylene(30) glyceryl monostearate, polyoxyethylene(20) sorbitan monooleate, sucrose fatty acid esters, hexaglycerin monooleate, hexaglycerin monolaurate, polyoxyethylene reduced lanolin, polyoxyethylene(20) lanolin alcohol, polyoxyethylene(25) glyceryl pyroglutamate isostearate diester, N acetylglutamine isostearyl ester, etc.
The microbicides may include, for example; trichlorohydroxydiphenyl ether, hinokitiol, tricrosan, chlorohexidine gluconate, phenoxyethanol, resorcinol, isopropylmethylphenol, azulene, salicylic acid, zinc pyrithione, benzalkonium chloride, photoreceptor No. 301, sodium mononitroguaiacol, etc.
The antioxidants may include, for example, butylhydroxyanisole, propyl gallate, and erysorbic acid.
The hormones may include, for example, ethynylestradiol, estrone, estradiol, etc.
The ultraviolet absorbents may include, for example, benzophenones such as dihydroxybenzophenone; as well as melanins, ethyl para-aminobenzoate, 2-ethylhexyl paradimethylaminobenzoate, cinoxate, 2-ethylhexyl paramethoxycinnamate, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanic acid, and fine particles of metal oxides, etc.
The anti-inflammatory agents may include, for example, dipotassium glycyrrhetinatel xcex2-glycyrrhetinic acid, allantoin, diphenhydramine hydrochloride, guaiazulehe, and 1-menthol, etc.
The refrigerants may include, for example, capsicum tincture, 1-menthol, etc.
The moisturizers may include, for example, L-pyrrolidonecarboxylic acid, sodium hyaluronate, chondroitin sulfate, etc.
The vitamins may include, for example, dl-xcex1-tocopherol acetate, dl-xcex1-tocopherol, d-xcex4-tocopherol, vitamin E, benzyl nicotinate, nicotinic acid amide, D-pantothenyl alcohol, pantothenylethyl ether, biotin, pyridoxine hydrochloride, riboflavin, etc.
The herb extracts may include, for example, Swertia herb extract, garlic extract, ginseng extract, aloe extract, cinchona extract, etc.
The tinctures may include, for example, capsicum tincture, ginger extract, cantharis tincture, etc.
Where the above-mentioned liquid-type preparations are used as spray, they may be combined with noninflammable gas or the like.
The vehicles for the solid-type preparations may include, for example, vaseline, solid paraffin, vegetable oil, mineral oil, lanolin, wax, and macrogol. To these may be added, if desired, any of the above-mentioned additives; emulsifiers such as lecithin; and lower alcohols such as ethyl alcohol, isopropyl alcohol, etc.
The amount of the hair-growing agent of the present invention to be applied varies, depending on the age, the body weight and the condition of the case to which it is applied, the curing effect of said agent, the mode of administration, the treating time, etc. For a hair tonic of the hair-growing agent of the present invention, for example, its percutaneous dose may be from 0.5 to 5 ml/adult, preferably from 1 to 3 ml/adult, and may be applied once or several times a day.